Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity.
Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 degrees C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD(+)-dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2-13 and up to 85 degrees C. We have determined the crystal structure of the enzyme-NAD(+) binary complex at 1.65A resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic-aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp-Gly-Gly sequence at positions 242-244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability.
Asada
Yukuhiko
Y
RIKEN SPring-8 Center, Harima Institute, Sayo-gun, Hyogo, Japan.
Endo
Satoshi
S
Inoue
Yukari
Y
Mamiya
Hiroaki
H
Hara
Akira
A
Kunishima
Naoki
N
Matsunaga
Toshiyuki
T
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003460:Crystallization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*
D002237:Carbohydrate Dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors